CN109694394B - Preparation method and application of tannin extract in sea buckthorn - Google Patents

Abstract

The invention discloses a preparation method and application of a tannin extract in sea buckthorn. The extract is prepared by soaking folium Hippophae in methanol to obtain extract; dispersing the extract with water, extracting with organic solvent, and recovering solvent under reduced pressure; dissolving the extract with 20% methanol, separating by Sephadex LH-20 column chromatography, gradient eluting with methanol-water (20:80 → 100:0) solvent system to obtain 4 parts Fr.1-4, and separating and purifying Fr.4(20g) by silica gel, MCI CHP-20 column chromatography and semi-preparative high performance liquid chromatography to obtain compound. The tannin extract has anti-inflammatory and anti-obesity activity, and can be used for preventing and treating inflammation and obesity.

Description

A kind of preparation method and use of tannin extract in sea buckthorn

technical field

The invention belongs to the field of traditional Chinese medicine extracts, and relates to an extract derived from sea buckthorn with anti-inflammatory and anti-obesity adjuvant therapy, in particular to a preparation method and use of a tannin extract from sea buckthorn.

Background technique

Obesity is mainly caused by eating too much and too little activity. The calorie intake is more than the calorie consumption, and the increase of fat synthesis is the material basis of obesity. Obesity due to fat metabolism disorder. Obese patients also need to continue medication to maintain their weight after using medication to lose weight. However, how to better apply these drugs in clinical practice remains to be explored. Drug use may cause drug side effects and drug resistance, so the selection of drug treatment indications must be very careful, and the decision should be made based on the patient's individual situation to weigh the possible benefits and potential risks.

Therefore, the status of traditional anti-obesity drugs has been reduced, and they are currently only used as short-term treatment measures. Nonetheless, this class of drugs remains an indispensable essential drug in the treatment of severely obese patients. Therefore, the search for anti-obesity drugs with less toxic and side effects is still of great significance for the treatment and prevention of obesity.

my country is rich in vegetation, which needs to be developed and applied urgently. Seabuckthorn (Hippophae rhamnoides L.) is a deciduous shrub or small tree of the genus Seabuckthorn in the family Elaeaceae, mainly distributed in countries along the Silk Road. my country is the country with the most abundant sea buckthorn germplasm resources and reserves in the world. There are 7 species and 7 subspecies, which are widely distributed in Northwest my country, North China, Northeast China, Southwest China and other places. Its reserves account for about 90% of the world's sea buckthorn resources.

SUMMARY OF THE INVENTION

The purpose of the present invention is to propose a preparation method and application of tannin extract in sea buckthorn.

In order to achieve the above object, the present invention is achieved through the following technical scheme: the use of the tannin extract from the sea buckthorn in an anti-obesity adjuvant therapeutic drug.

The tannin extract in the sea buckthorn has the components shown in formula 1, formula 2 and formula 3:

Wherein, H in R1, R2, R3, R4 and R5 is hydrogen or galloyl group.

The tannin extract in the sea buckthorn is prepared by the following method:

(1) 6kg of sea buckthorn leaf is soaked and extracted with 80% aqueous alcohol solution, filtered and merged extract, concentrated under reduced pressure to obtain extract 1.4kg, for subsequent use;

(2) after the standby extract is dispersed with water, extracted with an organic solvent, and the solvent is recovered under reduced pressure for use;

(3) Take the extract that has undergone the above steps, dissolve it in 20% methanol, carry out Sephadex LH-20 column chromatography, and elute with methanol-water (20:80→100:0) solvent system gradient to obtain 4 parts Fr.1-4, Fr.4 (20g) were separated and purified by silica gel, MCI CHP-20 column chromatography and semi-preparative high performance liquid chromatography to obtain compounds.

The aqueous alcohol solution is methanol, and the methanol is soaked and extracted repeatedly for 3-4 times, and the extraction temperature is 20-70 DEG C.

The organic solvent is chloroform, ethyl acetate and n-butanol.

The organic solvent extraction step is to extract 3-4 times with chloroform, ethyl acetate and n-butanol respectively, and the extraction temperature is 18-35°C.

The preparation method and application of the tannin extract of sea buckthorn according to the present invention has the beneficial effects that the tannin extract can be used for the prevention and treatment of obesity. The extract has a strong anti-obesity effect.

Instruction drawings

Figure 1 shows the inhibition rate of quercetin and most tannins on the accumulation and differentiation of TG in adipocytes.

Detailed ways

Example 1

1. Materials:

The sea buckthorn leaves used in the experiment were collected from Inner Mongolia and were dried leaves of Hippophae rhamnoides L..

2. Extraction and separation:

6 kg of sea buckthorn leaves were soaked and extracted with 80% methanol, repeated 3 times, filtered to combine the extracts, and concentrated under reduced pressure to obtain 1.4 kg of extract. After the extract was dispersed with water, it was extracted three times with chloroform, ethyl acetate and n-butanol, respectively, and the solvent was recovered under reduced pressure to obtain the corresponding extract, wherein the ethyl acetate layer and the water layer were 60 g and 933 g respectively. Take 57g of the extract from the ethyl acetate part, dissolve it in 20% methanol, carry out Sephadex LH-20 column chromatography, and elute with methanol-water (20:80→100:0) solvent system gradient to obtain 4 parts Fr.1 -4, Fr.4 (20g) was separated and purified by silica gel, MCI CHP-20 column chromatography and semi-preparative high performance liquid chromatography to obtain the compound.

3. Structure identification:

Compound 1: light brown powder, FAB-MS(m/z): 787[MH] ^- , HR-FAB-MS(m/z): found.787.0993[MH] ^- ( _{calcd.787.0992for C34H27O 22} ); ¹ H-NMR (Acetone-d ₆ , 600MHz) δ: 6.01 (1H, d, J=8.3Hz, glu, H-1), 4.09 (1H, t, J=8.6Hz, H-2) ,5.70(1H,t,J＝9.6Hz,H-3),5.51(1H,t,J＝9.6Hz,H-4),4.41(1H,m,H-5),4.58(1H,dd, J=12.4, 1.7Hz, H-6α), 4.49 (1H, dd, J=12.4, 5.2Hz, H-6β), 7.21, 7.15, 7.10, 7.07 (each 2H, s, galloyl); ¹³ C-NMR (Acetone-d ₆ , 150MHz)δ:94.8(glu,C-1),71.2(C-2),75.1(C-3),68.9(C-4),73.0(C-5),62.6(C -6),120.2,121.1,119.4,119.4(C-1,galloyl), 109.6,109.4,109.4(C-2,6),109.3,145.5,145.4,145.4,145.3(C-3,5),139.2 , 139.0, 138.5, 138.5 (C-4), 166.21, 166.2, 165.7, 165.3 (C=O).

Compound 2: light brown powder, FAB-MS(m/z): 787[MH] ^- , HR-FAB-MS(m/z): found.787.0993[MH] ^- ( _{calcd.787.0992for C34H27O 22} ); ¹ H-NMR (Acetone-d ₆ , 600MHz) δ: 6.19 (1H, d, J=8.6Hz, glu, H-1), 5.48 (1H, t, J=8.6Hz, H-2) , 5.69(1H,t,J＝8.9Hz,H-3),4.11(1H,m,H-4),4.16(1H,m,H-5),4.62(1H,dd,J＝12.1,1.8 Hz, H-6α), 4.58 (1H, dd, J=12.1, 4.7Hz, H-6 β), 7.19, 7.10, 7.09, 7.01 (each 2H, s, galloyl); ¹³ C-NMR (Acetone-d _6,150 MHz)δ: 93.5(glu,C-1), 71.8(C-2), 75.9(C-3), 68.4(C-4), 76.1(C-5), 63.7(C-6) ,121.6,121.4,120.8,120.1(C-1,galloyl),110.3,110.2,110.1, 110.0(C-2,6),146.1,146.1,145.9,145.9(C-3,5),139.7,139.2, 139.0, 138.9 (C-4), 166.6, 166.2, 165.8, 165.0 (C=O).

Compound 3: light brown powder FAB-MS (m/z): 939 [MH] ^- , HR-FAB-MS (m/z): found. 939.1103 [MH] ^- (calcd. 939.1102 for C ₄₁ H ₃₁ O ₂₆ ); ¹ H-NMR (Acetone-d ₆ , 600 MHz) δ: 6.34 (1H, d, J=8.5Hz, glu, H-1), 5.62 (1H, t, J=8.4Hz, H-2) , 6.02(1H,t,J=9.5Hz,H-3),5.67(1H,t,J=9.5Hz,H-4),4.58(1H,m,H-5),4.55(1H,dd, ^13C -NMR (Acetone-d ₆ , 150MHz) δ: 93.3 (glu, C-1), 71.8 (C-2), 73.3 (C-3), 69.3 (C-4), 74.0 (C-5), 62.8 (C-6), 121.4, 120.7, 120.6, 120.6, 119.9 (C-1, galloyl), 110.4, 110.3, 110.2, 110.1, 110.1 (C-2, 6), 146.1, 146.03, 145.98, 145.94, 145.86( C-3,5), 139.8, 139.4, 139.3, 139.2, 139.0 (C-4), 166.4, 165.9, 165.7, 165.6, 165.0 (C=O).

4. Evaluation of anti-obesity activity:

4.1 Evaluation of anti-obesity activity by inhibiting 3T3L-1 adipocyte triglyceride (TG) accumulation:

3T3-L1 adipocytes were cultured in high-glucose DMEM medium containing 10% newborn calf serum at 37°C and 5% CO ₂ . Induce differentiation medium (containing 10% fetal bovine serum, 10 μg/mL insulin, 1.0 μM dexamethasone, 500 μM 1-methyl-3-isobutylxanthine) and add different concentrations of samples to culture for 3 days, then switch to promoting Differentiation medium (containing 10% fetal bovine serum, 5 μg/mL insulin) and samples with different concentrations were added for 3 days, and the same medium was changed to the same medium and samples with different concentrations were added for 8 days. Taking the cell culture medium without sample as the negative control, observed under the microscope, it can be seen that the negative control cells have obvious fat droplets in the body; taking quercetin as the positive control. After 3T3-L1 adipocytes were washed with PBS(-), the cells were harvested with 25 mM Tris-HCl buffer containing 1 mM EDTA, homogenized by sonication, and the accumulated glycerol in the cells was determined using the LabAssayTM Triglyceride Kit (WAKO Pure Chemical Industries Ltd.). Triester (TG) content, GPDH activity was measured with glycerol phosphate dehydrogenase (GPDH) kit (TaKaRa BioInc.), respectively, quantitative analysis of fat accumulation and differentiation degree, evaluation of the anti-obesity activity of the test sample.

The experimental results are shown in Figure 1. At 10 μM concentration, the inhibition rate of positive control quercetin on adipocyte TG accumulation and differentiation is about 25%, and the inhibition rate of most tannins at the same concentration is about 30-40% , generally stronger than the inhibitory effect of quercetin.

Claims (4)

1. an extraction method of tannin extract in sea buckthorn, is characterized in that: The tannin extract in the sea buckthorn has the components shown in formula 1, formula 2 and formula 3:

Wherein, H in R1, R2, R3, R4, R5 is hydrogen; The tannin extract in the sea buckthorn is prepared by the following method: (1) 6kg of sea buckthorn leaf is soaked and extracted with 80% methanol, filtered and merged extract, concentrated under reduced pressure to obtain extract 1.4kg, for subsequent use; (2) after the standby extract is dispersed with water, extracted with an organic solvent, and the solvent is recovered under reduced pressure for use; (3) Take the extract after the above steps, dissolve it in 20% methanol, and carry out Sephadex LH-20 column chromatography, eluting with methanol-water 20:80→100:0 solvent system gradient to obtain 4 fractions of Fr. 1-4,Fr.4 20g was separated and purified by silica gel, MICHP-20 column chromatography and semi-preparative high performance liquid chromatography to obtain the compound. 2. A method for extracting tannin extract from sea buckthorn as claimed in claim 1, characterized in that: the methanol soaking is repeatedly extracted for 3-4 times, and the extraction temperature is 20-70 °C. 3. a kind of tannin extract extraction method in sea buckthorn as claimed in claim 2 is characterized in that: described organic solvent is chloroform, ethyl acetate, n-butanol. 4. a kind of tannin extract extraction method in sea buckthorn as claimed in claim 3, is characterized in that: described organic solvent extraction step is to extract 3-4 times with chloroform, ethyl acetate, n-butanol respectively, and extraction temperature is 18-35℃.

Images